Summary Melanoma is arguably the most virulent among human cancers, in part due to its propensity to metastasize, and its resistance to conventional anti-cancer therapies. One key factor responsible for treatment failure relates to tumor heterogeneity, particularly subpopulations that possess stem cell-like properties, known as melanoma initiating cells (MICs). Our long-term goal is to understand the molecular mechanisms, whereby MICs and their stroma collaborate to promote growth and progression, as a means to develop effective therapeutic strategies. In the current project, we focus on defining the microenvironmental niche, of CD133+/ABCB5+ MICs as a gateway to elucidate the complex cellular and molecular interplay that maintains the metabolic and replicative integrity of MICs. Our preliminary data indicate that : 1) the CD133+/ABCB5+ MICs are spatially arranged in a pattern identical to that of vasculogenic mimicry (VM); 2) the vessel-like channels so formed in VM are themselves CD144 (VE-cadherin)+ and intimately associated with authentic endothelial structures, in keeping with so-called perivascular niches; 3) melanoma cell expression of bone morphogenetic protein 7 (BMP7) and its antagonist Noggin is associated with tumor progression and also co-localizes to areas of VM; and 4) BMP7 upregulates the angiogenic factor VEGF in the stromal microenvironment, but at the same time induces selective apoptosis in Noggin-deficient melanoma cells. Collectively, our findings support the central hypothesis that CD133+/ABCB5+ MIC-associated BMP7 may not only support the maintenance of MICs by facilitating niche morphogenesis (through coordinated VM and angiogenesis), but also balance metabolic demands (through stimulating VEGF-dependent angiogenesis and inducing selective apoptosis in the competitive, Noggin-deficient, non-initiating population). Using multi-label immunofluorescence and immuno- guided laser capture microdissection followed by qRT-PCR, together with functional circulation analysis, we propose to further delineate the temporal/spatial relationship between melanoma cell CD133, ABCB5, BMP7 and CD144 expression, and the types/ patterns of microcirculation (e.g. melanoma VM channel formation vs. angiogenesis) in vivo (Aim 1a). The dynamics and functional impacts of CD133+ melanoma subsets in niche formation (Aim 1b) will be tested in an orthotopic xenograft model using inducible RNAi-mediated knockdown at varying tempos. Finally, to dissect the CD133+/ ABCB5+ MIC-associated BMP7 molecular signals in niche morphogenesis (Aim 2a) and tumor heterogeneity/ homeostasis (Aim 2b), we will employ loss- and gain-of- function approaches to assess the BMP7-VEGF/ BMP7-Noggin axes, in a tissue context in unique three- dimensional (3D) organotypic cultures in vitro and melanoma xenograft models in vivo. Defining mechanisms through which MIC-associated BMP7 signals contribute to niche development and maintenance offers a novel opportunity to therapeutically eliminate MICs directly or indirectly by targeting their stromal dependency.